Lipids for delivery of active agents

ABSTRACT

Compounds are provided having the following structure: Formula (I) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R, R 1 , R 2 , G 1 , G 2  and n are as defined herein. Use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, compositions comprising the compounds and methods for their use and preparation are also provided.

BACKGROUND Technical Field

The present invention generally relates to novel cationic lipids thatcan be used in combination with other lipid components, such as neutrallipids, cholesterol and polymer conjugated lipids, to form lipidnanoparticles with oligonucleotides, to facilitate the intracellulardelivery of therapeutic nucleic acids (e.g., oligonucleotides, messengerRNA) both in vitro and in vivo.

Description of the Related Art

There are many challenges associated with the delivery of nucleic acidsto affect a desired response in a biological system. Nucleic acid basedtherapeutics have enormous potential but there remains a need for moreeffective delivery of nucleic acids to appropriate sites within a cellor organism in order to realize this potential. Therapeutic nucleicacids include, e.g., messenger RNA (mRNA), antisense oligonucleotides,ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids,antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids,such as mRNA or plasmids, can be used to effect expression of specificcellular products as would be useful in the treatment of, for example,diseases related to a deficiency of a protein or enzyme. The therapeuticapplications of translatable nucleotide delivery are extremely broad asconstructs can be synthesized to produce any chosen protein sequence,whether or not indigenous to the system. The expression products of thenucleic acid can augment existing levels of protein, replace missing ornon-functional versions of a protein, or introduce new protein andassociated functionality in a cell or organism.

Some nucleic acids, such as miRNA inhibitors, can be used to effectexpression of specific cellular products that are regulated by miRNA aswould be useful in the treatment of, for example, diseases related todeficiency of protein or enzyme. The therapeutic applications of miRNAinhibition are extremely broad as constructs can be synthesized toinhibit one or more miRNA that would in turn regulate the expression ofmRNA products. The inhibition of endogenous miRNA can augment itsdownstream target endogenous protein expression and restore properfunction in a cell or organism as a means to treat disease associated toa specific miRNA or a group of miRNA.

Other nucleic acids can down-regulate intracellular levels of specificmRNA and, as a result, down-regulate the synthesis of the correspondingproteins through processes such as RNA interference (RNAi) orcomplementary binding of antisense RNA. The therapeutic applications ofantisense oligonucleotide and RNAi are also extremely broad, sinceoligonucleotide constructs can be synthesized with any nucleotidesequence directed against a target mRNA. Targets may include mRNAs fromnormal cells, mRNAs associated with disease-states, such as cancer, andmRNAs of infectious agents, such as viruses. To date, antisenseoligonucleotide constructs have shown the ability to specificallydown-regulate target proteins through degradation of the cognate mRNA inboth in vitro and in vivo models. In addition, antisense oligonucleotideconstructs are currently being evaluated in clinical studies.

However, two problems currently face the use of oligonucleotides intherapeutic contexts. First, free RNAs are susceptible to nucleasedigestion in plasma. Second, free RNAs have limited ability to gainaccess to the intracellular compartment where the relevant translationmachinery resides. Lipid nanoparticles formed from cationic lipids withother lipid components, such as neutral lipids, cholesterol, PEG,PEGylated lipids, and oligonucleotides have been used to blockdegradation of the RNAs in plasma and facilitate the cellular uptake ofthe oligonucleotides.

There remains a need for improved cationic lipids and lipidnanoparticles for the delivery of oligonucleotides. Preferably, theselipid nanoparticles would provide optimal drug:lipid ratios, protect thenucleic acid from degradation and clearance in serum, be suitable forsystemic or local delivery, and provide intracellular delivery of thenucleic acid. In addition, these lipid-nucleic acid particles should bewell-tolerated and provide an adequate therapeutic index, such thatpatient treatment at an effective dose of the nucleic acid is notassociated with unacceptable toxicity and/or risk to the patient. Thepresent invention provides these and related advantages.

BRIEF SUMMARY

In brief, the present invention provides lipid compounds, includingstereoisomers, pharmaceutically acceptable salts or tautomers thereof,which can be used alone or in combination with other lipid componentssuch as neutral lipids, charged lipids, steroids (including for example,all sterols) and/or their analogs, and/or polymer conjugated lipids toform lipid nanoparticles for the delivery of therapeutic agents. In someinstances, the lipid nanoparticles are used to deliver nucleic acidssuch as antisense and/or messenger RNA. Methods for use of such lipidnanoparticles for treatment of various diseases or conditions, such asthose caused by infectious entities and/or insufficiency of a protein,are also provided.

In one embodiment, compounds having the following structure (I) areprovided:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein R, R¹, R², G¹, G², and n are as defined herein.

Pharmaceutical compositions comprising one or more of the foregoingcompounds of structure (I) and a therapeutic agent are also provided. Insome embodiments, the pharmaceutical compositions further comprise oneor more components selected from neutral lipids, charged lipids,steroids and polymer conjugated lipids. Such compositions are useful forformation of lipid nanoparticles for the delivery of the therapeuticagent.

In other embodiments, the present invention provides a method foradministering a therapeutic agent to a patient in need thereof, themethod comprising preparing or providing a composition of lipidnanoparticles comprising the compound of structure (I) and a therapeuticagent and delivering or administering the composition to the patient.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand that theinvention may be practiced without these details.

The present invention is based, in part, upon the discovery of novelcationic (amino) lipids that provide advantages when used in lipidnanoparticles for the in vivo delivery of an active or therapeutic agentsuch as a nucleic acid into a cell of a mammal. In particular,embodiments of the present invention provide nucleic acid-lipidnanoparticle compositions comprising one or more of the novel cationiclipids described herein that provide increased activity of the nucleicacid and improved tolerability of the compositions in vivo, resulting ina significant increase in the therapeutic index as compared to nucleicacid-lipid nanoparticle compositions previously described. In otherembodiments, the disclosed lipids, and lipid nanoparticles comprisingthe same, have increased safety and/or tolerability when used fordelivery of active agents, such as nucleic acids.

In particular embodiments, the present invention provides novel cationiclipids that enable the formulation of improved compositions for the invitro and in vivo delivery of mRNA and/or other oligonucleotides. Insome embodiments, these improved lipid nanoparticle compositions areuseful for expression of protein encoded by mRNA. In other embodiments,these improved lipid nanoparticles compositions are useful forupregulation of endogenous protein expression by delivering miRNAinhibitors targeting one specific miRNA or a group of miRNA regulatingone target mRNA or several mRNA. In other embodiments, these improvedlipid nanoparticle compositions are useful for down-regulating (e.g.,silencing) the protein levels and/or mRNA levels of target genes. Insome other embodiments, the lipid nanoparticles are also useful fordelivery of mRNA and plasmids for expression of transgenes. In yet otherembodiments, the lipid nanoparticle compositions are useful for inducinga pharmacological effect resulting from expression of a protein, e.g.,increased production of red blood cells through the delivery of asuitable erythropoietin mRNA, or protection against infection throughdelivery of mRNA encoding for a suitable antigen or antibody.

The lipid nanoparticles and compositions of embodiments of the presentinvention may be used for a variety of purposes, including the deliveryof encapsulated or associated (e.g., complexed) therapeutic agents suchas nucleic acids to cells, both in vitro and in vivo. Accordingly,embodiments of the present invention provide methods of treating orpreventing diseases or disorders in a subject in need thereof bycontacting the subject with a lipid nanoparticle that encapsulates or isassociated with a suitable therapeutic agent, wherein the lipidnanoparticle comprises one or more of the novel cationic lipidsdescribed herein.

As described herein, embodiments of the lipid nanoparticles of thepresent invention are particularly useful for the delivery of nucleicacids, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA,microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs),messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalentRNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, thelipid nanoparticles and compositions of certain embodiments of thepresent invention may be used to induce expression of a desired proteinboth in vitro and in vivo by contacting cells with a lipid nanoparticlecomprising one or more novel cationic lipids described herein, whereinthe lipid nanoparticle encapsulates or is associated with a nucleic acidthat is expressed to produce the desired protein (e.g., a messenger RNAor plasmid encoding the desired protein) or inhibit processes thatterminate expression of mRNA (e.g., miRNA inhibitors). Alternatively,the lipid nanoparticles and compositions of embodiments of the presentinvention may be used to decrease the expression of target genes andproteins both in vitro and in vivo by contacting cells with a lipidnanoparticle comprising one or more novel cationic lipids describedherein, wherein the lipid nanoparticle encapsulates or is associatedwith a nucleic acid that reduces target gene expression (e.g., anantisense oligonucleotide or small interfering RNA (siRNA)). The lipidnanoparticles and compositions of embodiments of the present inventionmay also be used for co-delivery of different nucleic acids (e.g. mRNAand plasmid DNA) separately or in combination, such as may be useful toprovide an effect requiring colocalization of different nucleic acids(e.g. mRNA encoding for a suitable gene modifying enzyme and DNAsegment(s) for incorporation into the host genome).

Nucleic acids for use with embodiments of this invention may be preparedaccording to any available technique. For mRNA, the primary methodologyof preparation is, but not limited to, enzymatic synthesis (also termedin vitro transcription) which currently represents the most efficientmethod to produce long sequence-specific mRNA. In vitro transcriptiondescribes a process of template-directed synthesis of RNA molecules froman engineered DNA template comprised of an upstream bacteriophagepromoter sequence (e.g., including but not limited to that from the T7,T3 and SP6 coliphage) linked to a downstream sequence encoding the geneof interest. Template DNA can be prepared for in vitro transcriptionfrom a number of sources with appropriate techniques which are wellknown in the art including, but not limited to, plasmid DNA andpolymerase chain reaction amplification (see Linpinsel, J. L and Conn,G. L., General protocols for preparation of plasmid DNA template andBowman, J. C., Azizi, B., Lenz, T. K., Ray, P., and Williams, L. D. inRNA in vitro transcription and RNA purification by denaturing PAGE inRecombinant and in vitro RNA syntheses Methods v. 941 Conn G. L. (ed),New York, N.Y. Humana Press, 2012)

Transcription of the RNA occurs in vitro using the linearized DNAtemplate in the presence of the corresponding RNA polymerase andadenosine, guanosine, uridine and cytidine ribonucleoside triphosphates(rNTPs) under conditions that support polymerase activity whileminimizing potential degradation of the resultant mRNA transcripts. Invitro transcription can be performed using a variety of commerciallyavailable kits including, but not limited to RiboMax Large Scale RNAProduction System (Promega), MegaScript Transcription kits (LifeTechnologies) as well as with commercially available reagents includingRNA polymerases and rNTPs. The methodology for in vitro transcription ofmRNA is well known in the art. (see, e.g. Losick, R., 1972, In vitrotranscription, Ann Rev Biochem v. 41 409-46; Kamakaka, R. T. and Kraus,W. L. 2001. In Vitro Transcription. Current Protocols in Cell Biology.2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis ofRNA by In Vitro Transcription in RNA in Methods in Molecular Biology v.703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J. L.and Green, R., 2013, Chapter Five—In vitro transcription from plasmid orPCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of whichare incorporated herein by reference).

The desired in vitro transcribed mRNA is then purified from theundesired components of the transcription or associated reactions(including unincorporated rNTPs, protein enzyme, salts, short RNAoligos, etc.). Techniques for the isolation of the mRNA transcripts arewell known in the art. Well known procedures include phenol/chloroformextraction or precipitation with either alcohol (ethanol, isopropanol)in the presence of monovalent cations or lithium chloride. Additional,non-limiting examples of purification procedures which can be usedinclude size exclusion chromatography (Lukavsky, P. J. and Puglisi, J.D., 2004, Large-scale preparation and purification ofpolyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893),silica-based affinity chromatography and polyacrylamide gelelectrophoresis (Bowman, J. C., Azizi, B., Lenz, T. K., Ray, P., andWilliams, L. D. in RNA in vitro transcription and RNA purification bydenaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941Conn G. L. (ed), New York, N.Y. Humana Press, 2012). Purification can beperformed using a variety of commercially available kits including, butnot limited to SV Total Isolation System (Promega) and In VitroTranscription Cleanup and Concentration Kit (Norgen Biotek).

Furthermore, while reverse transcription can yield large quantities ofmRNA, the products can contain a number of aberrant RNA impuritiesassociated with undesired polymerase activity which may need to beremoved from the full-length mRNA preparation. These include short RNAsthat result from abortive transcription initiation as well asdouble-stranded RNA (dsRNA) generated by RNA-dependent RNA polymeraseactivity, RNA-primed transcription from RNA templates andself-complementary 3′ extension. It has been demonstrated that thesecontaminants with dsRNA structures can lead to undesiredimmunostimulatory activity through interaction with various innateimmune sensors in eukaryotic cells that function to recognize specificnucleic acid structures and induce potent immune responses. This inturn, can dramatically reduce mRNA translation since protein synthesisis reduced during the innate cellular immune response. Therefore,additional techniques to remove these dsRNA contaminants have beendeveloped and are known in the art including but not limited toscaleable HPLC purification (see, e.g., Kariko, K., Muramatsu, H.,Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA fortherapy: HPLC purification eliminates immune activation and improvestranslation of nucleoside-modified, protein-encoding mRNA, Nucl AcidRes, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K.,HPLC Purification of in vitro transcribed long RNA in SyntheticMessenger RNA and Cell Metabolism Modulation in Methods in MolecularBiology v.969 (Rabinovich, P. H. Ed), 2013). HPLC purified mRNA has beenreported to be translated at much greater levels, particularly inprimary cells and in vivo.

A significant variety of modifications have been described in the artwhich are used to alter specific properties of in vitro transcribedmRNA, and improve its utility. These include, but are not limited tomodifications to the 5′ and 3′ termini of the mRNA. Endogenouseukaryotic mRNA typically contain a cap structure on the 5′-end of amature molecule which plays an important role in mediating binding ofthe mRNA Cap Binding Protein (CBP), which is in turn responsible forenhancing mRNA stability in the cell and efficiency of mRNA translation.Therefore, highest levels of protein expression are achieved with cappedmRNA transcripts. The 5′-cap contains a 5′-5′-triphosphate linkagebetween the 5′-most nucleotide and guanine nucleotide. The conjugatedguanine nucleotide is methylated at the N7 position. Additionalmodifications include methylation of the ultimate and penultimate most5′-nucleotides on the 2′-hydroxyl group.

Multiple distinct cap structures can be used to generate the 5′-cap ofin vitro transcribed synthetic mRNA. 5′-capping of synthetic mRNA can beperformed co-transcriptionally with chemical cap analogs (i.e., cappingduring in vitro transcription). For example, the Anti-Reverse Cap Analog(ARCA) cap contains a 5′-5′-triphosphate guanine-guanine linkage whereone guanine contains an N7 methyl group as well as a 3′-O-methyl group.However, up to 20% of transcripts remain uncapped during thisco-transcriptional process and the synthetic cap analog is not identicalto the 5′-cap structure of an authentic cellular mRNA, potentiallyreducing translatability and cellular stability. Alternatively,synthetic mRNA molecules may also be enzymatically cappedpost-transcriptionally. These may generate a more authentic 5′-capstructure that more closely mimics, either structurally or functionally,the endogenous 5′-cap which have enhanced binding of cap bindingproteins, increased half-life and reduced susceptibility to 5′endonucleases and/or reduced 5′ decapping. Numerous synthetic 5′-capanalogs have been developed and are known in the art to enhance mRNAstability and translatability (see, e.g., Grudzien-Nogalska, E.,Kowalska, J., Su, W., Kuhn, A. N., Slepenkov, S. V., Darynkiewicz, E.,Sahin, U., Jemielity, J., and Rhoads, R. E., Synthetic mRNAs withsuperior translation and stability properties in Synthetic Messenger RNAand Cell Metabolism Modulation in Methods in Molecular Biology v.969(Rabinovich, P. H. Ed), 2013).

On the 3′-terminus, a long chain of adenine nucleotides (poly-A tail) isnormally added to mRNA molecules during RNA processing. Immediatelyafter transcription, the 3′ end of the transcript is cleaved to free a3′ hydroxyl to which poly-A polymerase adds a chain of adeninenucleotides to the RNA in a process called polyadenylation. The poly-Atail has been extensively shown to enhance both translational efficiencyand stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A),poly (A) binding protein and the regulation of mRNA stability, TrendsBio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001, Regulationof mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M.And Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard ineukaryotes, scavenger in bacteria, Cell, v.111, 611-613).

Poly (A) tailing of in vitro transcribed mRNA can be achieved usingvarious approaches including, but not limited to, cloning of a poly (T)tract into the DNA template or by post-transcriptional addition usingPoly (A) polymerase. The first case allows in vitro transcription ofmRNA with poly (A) tails of defined length, depending on the size of thepoly (T) tract, but requires additional manipulation of the template.The latter case involves the enzymatic addition of a poly (A) tail to invitro transcribed mRNA using poly (A) polymerase which catalyzes theincorporation of adenine residues onto the 3′termini of RNA, requiringno additional manipulation of the DNA template, but results in mRNA withpoly(A) tails of heterogeneous length. 5′-capping and 3′-poly (A)tailing can be performed using a variety of commercially available kitsincluding, but not limited to Poly (A) Polymerase Tailing kit(EpiCenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit(Life Technologies) as well as with commercially available reagents,various ARCA caps, Poly (A) polymerase, etc.

In addition to 5′ cap and 3′ poly adenylation, other modifications ofthe in vitro transcripts have been reported to provide benefits asrelated to efficiency of translation and stability. It is well known inthe art that pathogenic DNA and RNA can be recognized by a variety ofsensors within eukaryotes and trigger potent innate immune responses.The ability to discriminate between pathogenic and self DNA and RNA hasbeen shown to be based, at least in part, on structure and nucleosidemodifications since most nucleic acids from natural sources containmodified nucleosides. In contrast, in vitro synthesized RNA lacks thesemodifications, thus rendering it immunostimulatory which in turn caninhibit effective mRNA translation as outlined above. The introductionof modified nucleosides into in vitro transcribed mRNA can be used toprevent recognition and activation of RNA sensors, thus mitigating thisundesired immunostimulatory activity and enhancing translation capacity(see, e.g., Kariko, K. And Weissman, D. 2007, Naturally occurringnucleoside modifications suppress the immunostimulatory activity of RNA:implication for therapeutic RNA development, Curr Opin Drug DiscovDevel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K.,In vitro transcription of long RNA containing modified nucleosides inSynthetic Messenger RNA and Cell Metabolism Modulation in Methods inMolecular Biology v.969 (Rabinovich, P. H. Ed), 2013; Kariko, K.,Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., Weissman,D., 2008, Incorporation of Pseudouridine Into mRNA Yields SuperiorNonimmunogenic Vector With Increased Translational Capacity andBiological Stability, Mol Ther v.16, 1833-1840). The modifiednucleosides and nucleotides used in the synthesis of modified RNAs canbe prepared monitored and utilized using general methods and proceduresknown in the art. A large variety of nucleoside modifications areavailable that may be incorporated alone or in combination with othermodified nucleosides to some extent into the in vitro transcribed mRNA(see, e.g., US2012/0251618). In vitro synthesis of nucleoside-modifiedmRNA has been reported to have reduced ability to activate immunesensors with a concomitant enhanced translational capacity.

Other components of mRNA which can be modified to provide benefit interms of translatability and stability include the 5′ and 3′untranslated regions (UTR). Optimization of the UTRs (favorable 5′ and3′ UTRs can be obtained from cellular or viral RNAs), either both orindependently, have been shown to increase mRNA stability andtranslational efficiency of in vitro transcribed mRNA (see, e.g., Pardi,N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription oflong RNA containing modified nucleosides in Synthetic Messenger RNA andCell Metabolism Modulation in Methods in Molecular Biology v.969(Rabinovich, P. H. Ed), 2013).

In addition to mRNA, other nucleic acid payloads may be used for thisinvention. For oligonucleotides, methods of preparation include but arenot limited to chemical synthesis and enzymatic, chemical cleavage of alonger precursor, in vitro transcription as described above, etc.Methods of synthesizing DNA and RNA nucleotides are widely used and wellknown in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotidesynthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.:IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis:methods and applications, Methods in Molecular Biology, v. 288 (Clifton,N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporatedherein by reference).

For plasmid DNA, preparation for use with embodiments of this inventioncommonly utilizes, but is not limited to, expansion and isolation of theplasmid DNA in vitro in a liquid culture of bacteria containing theplasmid of interest. The presence of a gene in the plasmid of interestthat encodes resistance to a particular antibiotic (penicillin,kanamycin, etc.) allows those bacteria containing the plasmid ofinterest to selectively grow in antibiotic-containing cultures. Methodsof isolating plasmid DNA are widely used and well known in the art (see,e.g., Heilig, J., Elbing, K. L. and Brent, R., (2001), Large-ScalePreparation of Plasmid DNA, Current Protocols in Molecular Biology,41:II:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstrom, S.,Björnestedt, R. and Schmidt, S. R., (2008), Large-scale production ofendotoxin-free plasmids for transient expression in mammalian cellculture, Biotechnol. Bioeng., 99: 557-566; and U.S. Pat. No. 6,197,553B1). Plasmid isolation can be performed using a variety of commerciallyavailable kits including, but not limited to Plasmid Plus (Qiagen),GenJET plasmid MaxiPrep (Thermo) and PureYield MaxiPrep (Promega) kitsas well as with commercially available reagents.

Various exemplary embodiments of the cationic lipids of the presentinvention, lipid nanoparticles and compositions comprising the same, andtheir use to deliver active (e.g., therapeutic agents), such as nucleicacids, to modulate gene and protein expression, are described in furtherdetail below.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open andinclusive sense, that is, as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. As used in the specification andclaims, the singular form “a”, “an” and “the” include plural referencesunless the context clearly dictates otherwise.

The phrase “induce expression of a desired protein” refers to theability of a nucleic acid to increase expression of the desired protein.To examine the extent of protein expression, a test sample (e.g., asample of cells in culture expressing the desired protein) or a testmammal (e.g., a mammal such as a human or an animal) model such as arodent (e.g., mouse) or a non-human primate (e.g., monkey) model iscontacted with a nucleic acid (e.g., nucleic acid in combination with alipid of the present invention). Expression of the desired protein inthe test sample or test animal is compared to expression of the desiredprotein in a control sample (e.g., a sample of cells in cultureexpressing the desired protein) or a control mammal (e.g., a mammal suchas a human or an animal) model such as a rodent (e.g., mouse) ornon-human primate (e.g., monkey) model that is not contacted with oradministered the nucleic acid. When the desired protein is present in acontrol sample or a control mammal, the expression of a desired proteinin a control sample or a control mammal may be assigned a value of 1.0.In particular embodiments, inducing expression of a desired protein isachieved when the ratio of desired protein expression in the test sampleor the test mammal to the level of desired protein expression in thecontrol sample or the control mammal is greater than 1, for example,about 1.1, 1.5, 2.0. 5.0 or 10.0. When a desired protein is not presentin a control sample or a control mammal, inducing expression of adesired protein is achieved when any measurable level of the desiredprotein in the test sample or the test mammal is detected. One ofordinary skill in the art will understand appropriate assays todetermine the level of protein expression in a sample, for example dotblots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, and phenotypic assays, or assaysbased on reporter proteins that can produce fluorescence or luminescenceunder appropriate conditions.

The phrase “inhibiting expression of a target gene” refers to theability of a nucleic acid to silence, reduce, or inhibit the expressionof a target gene. To examine the extent of gene silencing, a test sample(e.g., a sample of cells in culture expressing the target gene) or atest mammal (e.g., a mammal such as a human or an animal) model such asa rodent (e.g., mouse) or a non-human primate (e.g., monkey) model iscontacted with a nucleic acid that silences, reduces, or inhibitsexpression of the target gene. Expression of the target gene in the testsample or test animal is compared to expression of the target gene in acontrol sample (e.g., a sample of cells in culture expressing the targetgene) or a control mammal (e.g., a mammal such as a human or an animal)model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey)model that is not contacted with or administered the nucleic acid. Theexpression of the target gene in a control sample or a control mammalmay be assigned a value of 100%. In particular embodiments, silencing,inhibition, or reduction of expression of a target gene is achieved whenthe level of target gene expression in the test sample or the testmammal relative to the level of target gene expression in the controlsample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Inother words, the nucleic acids are capable of silencing, reducing, orinhibiting the expression of a target gene by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% in a test sample or a test mammal relative to thelevel of target gene expression in a control sample or a control mammalnot contacted with or administered the nucleic acid. Suitable assays fordetermining the level of target gene expression include, withoutlimitation, examination of protein or mRNA levels using techniques knownto those of skill in the art, such as, e.g., dot blots, northern blots,in situ hybridization, ELISA, immunoprecipitation, enzyme function, aswell as phenotypic assays known to those of skill in the art.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as a therapeutic nucleic acid is anamount sufficient to produce the desired effect, e.g., an increase orinhibition of expression of a target sequence in comparison to thenormal expression level detected in the absence of the nucleic acid. Anincrease in expression of a target sequence is achieved when anymeasurable level is detected in the case of an expression product thatis not present in the absence of the nucleic acid. In the case where theexpression product is present at some level prior to contact with thenucleic acid, an in increase in expression is achieved when the foldincrease in value obtained with a nucleic acid such as mRNA relative tocontrol is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000,5000, 10000 or greater. Inhibition of expression of a target gene ortarget sequence is achieved when the value obtained with a nucleic acidsuch as antisense oligonucleotide relative to the control is about 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of atarget gene or target sequence include, e.g., examination of protein orRNA levels using techniques known to those of skill in the art such asdot blots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, fluorescence or luminescence ofsuitable reporter proteins, as well as phenotypic assays known to thoseof skill in the art.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA, RNA, and hybrids thereof. DNA maybe in the form of antisense molecules, plasmid DNA, cDNA, PCR products,or vectors. RNA may be in the form of small hairpin RNA (shRNA),messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA,dicer substrate RNA or viral RNA (vRNA), and combinations thereof.Nucleic acids include nucleic acids containing known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, and which have similarbinding properties as the reference nucleic acid. Examples of suchanalogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, single nucleotide polymorphisms, andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka etal., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell.Probes, 8:91-98 (1994)). “Nucleotides” contain a sugar deoxyribose (DNA)or ribose (RNA), a base, and a phosphate group. Nucleotides are linkedtogether through the phosphate groups. “Bases” include purines andpyrimidines, which further include natural compounds adenine, thymine,guanine, cytosine, uracil, inosine, and natural analogs, and syntheticderivatives of purines and pyrimidines, which include, but are notlimited to, modifications which place new reactive groups such as, butnot limited to, amines, alcohols, thiols, carboxylates, andalkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are generallycharacterized by being poorly soluble in water, but soluble in manyorganic solvents. They are usually divided into at least three classes:(1) “simple lipids,” which include fats and oils as well as waxes; (2)“compound lipids,” which include phospholipids and glycolipids; and (3)“derived lipids” such as steroids.

A “steroid” is a compound comprising the following carbon skeleton:

Non-limiting examples of steroids include cholesterol, and the like.

A “cationic lipid” refers to a lipid capable of being positivelycharged. Exemplary cationic lipids include one or more amine group(s)which bear the positive charge. Preferred cationic lipids are ionizablesuch that they can exist in a positively charged or neutral formdepending on pH. The ionization of the cationic lipid affects thesurface charge of the lipid nanoparticle under different pH conditions.This charge state can influence plasma protein absorption, bloodclearance and tissue distribution (Semple, S. C., et al., Adv. DrugDeliv Rev 32:3-17 (1998)) as well as the ability to form endosomolyticnon-bilayer structures (Hafez, I. M., et al., Gene Ther 8:1188-1196(2001)) critical to the intracellular delivery of nucleic acids.

The term “polymer conjugated lipid” refers to a molecule comprising botha lipid portion and a polymer portion. An example of a polymerconjugated lipid is a pegylated lipid. The term “pegylated lipid” refersto a molecule comprising both a lipid portion and a polyethylene glycolportion. Pegylated lipids are known in the art and include1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) andthe like.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, but are not limited to,phosphotidylcholines such as 1,2-Distearoyl-sn-glycero-3-phosphocholine(DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),phophatidylethanolamines such as1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins(SM), ceramides, steroids such as sterols and their derivatives. Neutrallipids may be synthetic or naturally derived.

The term “charged lipid” refers to any of a number of lipid species thatexist in either a positively charged or negatively charged formindependent of the pH within a useful physiological range, e.g., pH −3to pH −9. Charged lipids may be synthetic or naturally derived. Examplesof charged lipids include phosphatidylserines, phosphatidic acids,phosphatidylglycerols, phosphatidylinositols, sterol hemisuccinates,dialkyl trimethylammonium-propanes, (e.g., DOTAP, DOTMA), dialkyldimethylaminopropanes, ethyl phosphocholines, dimethylaminoethanecarbamoyl sterols (e.g., DC-Chol).

The term “lipid nanoparticle” refers to particles having at least onedimension on the order of nanometers (e.g., 1-1,000 nm) which includeone or more of the compounds of structure (I) or other specifiedcationic lipids. In some embodiments, lipid nanoparticles comprising thedisclosed cationic lipids (e.g., compounds of structure (I)) areincluded in a formulation that can be used to deliver an active agent ortherapeutic agent, such as a nucleic acid (e.g., mRNA) to a target siteof interest (e.g., cell, tissue, organ, tumor, and the like). In someembodiments, the lipid nanoparticles comprise a compound of structure(I) and a nucleic acid. Such lipid nanoparticles typically comprise acompound of structure (I) and one or more excipient selected fromneutral lipids, charged lipids, steroids and polymer conjugated lipids.In some embodiments, the active agent or therapeutic agent, such as anucleic acid, may be encapsulated in the lipid portion of the lipidnanoparticle or an aqueous space enveloped by some or all of the lipidportion of the lipid nanoparticle, thereby protecting it from enzymaticdegradation or other undesirable effects induced by the mechanisms ofthe host organism or cells, e.g., an adverse immune response.

In various embodiments, the lipid nanoparticles have a mean diameter offrom about 30 nm to about 150 nm, from about 40 nm to about 150 nm, fromabout 50 nm to about 150 nm, from about 60 nm to about 130 nm, fromabout 70 nm to about 110 nm, from about 70 nm to about 100 nm, fromabout 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In certain embodiments, nucleic acids,when present in the lipid nanoparticles, are resistant in aqueoussolution to degradation with a nuclease. Lipid nanoparticles comprisingnucleic acids and their method of preparation are disclosed in, e.g.,U.S. Patent Publication Nos. 2004/0142025, 2007/0042031 and PCT Pub.Nos. WO 2013/016058 and WO 2013/086373, the full disclosures of whichare herein incorporated by reference in their entirety for all purposes.

As used herein, “lipid encapsulated” refers to a lipid nanoparticle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., mRNA), with full encapsulation, partial encapsulation, or both.In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated inthe lipid nanoparticle.

As used herein, the term “aqueous solution” refers to a compositioncomprising water.

“Serum-stable” in relation to nucleic acid-lipid nanoparticles meansthat the nucleotide is not significantly degraded after exposure to aserum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of a therapeuticproduct that can result in a broad exposure of an active agent within anorganism. Some techniques of administration can lead to the systemicdelivery of certain agents, but not others. Systemic delivery means thata useful, preferably therapeutic, amount of an agent is exposed to mostparts of the body. Systemic delivery of lipid nanoparticles can be byany means known in the art including, for example, intravenous,intraarterial, subcutaneous, and intraperitoneal delivery. In someembodiments, systemic delivery of lipid nanoparticles is by intravenousdelivery.

“Local delivery,” as used herein, refers to delivery of an active agentdirectly to a target site within an organism. For example, an agent canbe locally delivered by direct injection into a disease site such as atumor, other target site such as a site of inflammation, or a targetorgan such as the liver, heart, pancreas, kidney, and the like. Localdelivery can also include topical applications or localized injectiontechniques such as intramuscular, subcutaneous or intradermal injection.Local delivery does not preclude a systemic pharmacological effect.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double (alkenyl) and/or triplebonds (alkynyl)), having, for example, from one to twenty-four carbonatoms (C₁-C₂₄ alkyl), four to twenty carbon atoms (C₄-C₂₀ alkyl), six tosixteen carbon atoms (C₆-C₁₆ alkyl), six to nine carbon atoms (C₆-C₉alkyl), one to fifteen carbon atoms (C₁-C₁₅ alkyl), one to twelve carbonatoms (C₁-C₁₂ alkyl), one to eight carbon atoms (C₁-C₅ alkyl) or one tosix carbon atoms (C₁-C₆ alkyl) and which is attached to the rest of themolecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl(iso propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl),3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl,pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl,hexynyl, and the like. Unless stated otherwise specifically in thespecification, an alkyl group is optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double (alkenylene) and/ortriple bonds (alkynylene)), and having, for example, from one totwenty-four carbon atoms (C₁-C₂₄ alkylene), one to fifteen carbon atoms(C₁-C₁₅ alkylene), one to twelve carbon atoms (C₁-C₁₂ alkylene), one toeight carbon atoms (C₁-C₈ alkylene), one to six carbon atoms (C₁-C₆alkylene), two to four carbon atoms (C₂-C₄ alkylene), one to two carbonatoms (C₁-C₂ alkylene), e.g., methylene, ethylene, propylene,n-butylene, ethenylene, propenylene, n-butenylene, propynylene,n-butynylene, and the like. The alkylene chain is attached to the restof the molecule through a single or double bond and to the radical groupthrough a single or double bond. The points of attachment of thealkylene chain to the rest of the molecule and to the radical group canbe through one carbon or any two carbons within the chain. Unless statedotherwise specifically in the specification, an alkylene chain may beoptionally substituted.

“Cycloalkyl” or “carbocyclic ring” refers to a stable non-aromaticmonocyclic or polycyclic hydrocarbon radical consisting solely of carbonand hydrogen atoms, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic radicalsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example,adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl,and the like. Unless otherwise stated specifically in the specification,a cycloalkyl group may be optionally substituted.

“Cycloalkylene” is a divalent cycloalkyl group. Unless otherwise statedspecifically in the specification, a cycloalkylene group may beoptionally substituted.

The term “substituted” used herein means any of the above groups (e.g.,alkyl, alkylene, cycloalkyl or cycloalkylene) wherein at least onehydrogen atom is replaced by a bond to a non-hydrogen atom such as, butnot limited to: a halogen atom such as F, Cl, Br, or I; oxo groups (═O);hydroxyl groups (—OH); C₁-C₁₂ alkyl groups; cycloalkyl groups;—(C═O)OR′; —O(C═O)R′; —C(═O)R′; —OR′; —S(O)_(x)R′; —S—SR′; —C(═O)SR′;—SC(═O)R′; —NR′R′; —NR′C(═O)R′; —C(═O)NR′R′; —NR′C(═O)NR′R′;—OC(═O)NR′R′; —NR′C(═O)OR′; —NR′S(O)_(x)NR′R′; —NR′S(O)_(x)R′; and—S(O)_(x)NR′R′, wherein: R′ is, at each occurrence, independently H,C₁-C₁₅ alkyl or cycloalkyl, and x is 0, 1 or 2. In some embodiments thesubstituent is a C₁-C₁₂ alkyl group. In other embodiments, thesubstituent is a cycloalkyl group. In other embodiments, the substituentis a halo group, such as fluoro. In other embodiments, the substituentis an oxo group. In other embodiments, the substituent is a hydroxylgroup. In other embodiments, the substituent is an alkoxy group (—OR′).In other embodiments, the substituent is a carboxyl group. In otherembodiments, the substituent is an amine group (—NR′R′).

“Optional” or “optionally” (e.g., optionally substituted) means that thesubsequently described event of circumstances may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances in which it does not. For example, “optionallysubstituted alkyl” means that the alkyl radical may or may not besubstituted and that the description includes both substituted alkylradicals and alkyl radicals having no substitution.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound of the invention. Thus, the term “prodrug” refers to ametabolic precursor of a compound of the invention that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject in need thereof, but is converted in vivo to an activecompound of the invention. Prodrugs are typically rapidly transformed invivo to yield the parent compound of the invention, for example, byhydrolysis in blood. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in a mammalianorganism (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24(Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi,T., et al., A.C.S. Symposium Series, Vol. 14, and in BioreversibleCarriers in Drug Design, Ed. Edward B. Roche, American PharmaceuticalAssociation and Pergamon Press, 1987.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound of the invention in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound of the invention may be prepared by modifying functional groupspresent in the compound of the invention in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound of the invention. Prodrugs include compounds of theinvention wherein a hydroxy, amino or mercapto group is bonded to anygroup that, when the prodrug of the compound of the invention isadministered to a mammalian subject, cleaves to form a free hydroxy,free amino or free mercapto group, respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol or amide derivatives of amine functional groupsin the compounds of the invention and the like.

The invention disclosed herein is also meant to encompass allpharmaceutically acceptable compounds of the compound of structure (I)being isotopically-labelled by having one or more atoms replaced by anatom having a different atomic mass or mass number. Examples of isotopesthat can be incorporated into the disclosed compounds include isotopesof hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine,and iodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P,³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I, respectively. These radiolabeledcompounds could be useful to help determine or measure the effectivenessof the compounds, by characterizing, for example, the site or mode ofaction, or binding affinity to pharmacologically important site ofaction. Certain isotopically-labelled compounds of structure (I), (IA)or (IB), for example, those incorporating a radioactive isotope, areuseful in drug and/or substrate tissue distribution studies. Theradioactive isotopes tritium, i.e., ³H, and carbon-14, i.e., ¹⁴C, areparticularly useful for this purpose in view of their ease ofincorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e., ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled compoundsof structure (I) can generally be prepared by conventional techniquesknown to those skilled in the art or by processes analogous to thosedescribed in the Preparations and Examples as set out below using anappropriate isotopically-labeled reagent in place of the non-labeledreagent previously employed.

Embodiments of the invention disclosed herein is also meant to encompassthe in vivo metabolic products of the disclosed compounds. Such productsmay result from, for example, the oxidation, reduction, hydrolysis,amidation, esterification, and the like of the administered compound,primarily due to enzymatic processes. Accordingly, embodiments of theinvention include compounds produced by a process comprisingadministering a compound of this invention to a mammal for a period oftime sufficient to yield a metabolic product thereof. Such products aretypically identified by administering a radiolabeled compound of theinvention in a detectable dose to an animal, such as rat, mouse, guineapig, monkey, or to human, allowing sufficient time for metabolism tooccur, and isolating its conversion products from the urine, blood orother biological samples.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Mammal” includes humans and both domestic animals such as laboratoryanimals and household pets (e.g., cats, dogs, swine, cattle, sheep,goats, horses, rabbits), and non-domestic animals such as wildlife andthe like.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of a compound of the invention(i.e., a compound of structure (I)). As used herein, the term “solvate”refers to an aggregate that comprises one or more molecules of acompound of the invention with one or more molecules of solvent. Thesolvent may be water, in which case the solvate may be a hydrate.Alternatively, the solvent may be an organic solvent. Thus, thecompounds of the present invention may exist as a hydrate, including amonohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate,tetrahydrate and the like, as well as the corresponding solvated forms.Solvates of compound of the invention may be true solvates, while inother cases, the compound of the invention may merely retainadventitious water or be a mixture of water plus some adventitioussolvent.

A “pharmaceutical composition” refers to a formulation of a compound ofthe invention and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

“Effective amount” or “therapeutically effective amount” refers to thatamount of a compound of the invention which, when administered to amammal, preferably a human, is sufficient to effect treatment in themammal, preferably a human. The amount of a lipid nanoparticle of theinvention which constitutes a “therapeutically effective amount” willvary depending on the compound, the condition and its severity, themanner of administration, and the age of the mammal to be treated, butcan be determined routinely by one of ordinary skill in the art havingregard to his own knowledge and to this disclosure.

“Treating” or “treatment” as used herein covers the treatment of thedisease or condition of interest in a mammal, preferably a human, havingthe disease or condition of interest, and includes:

(i) preventing the disease or condition from occurring in a mammal, inparticular, when such mammal is predisposed to the condition but has notyet been diagnosed as having it;

(ii) inhibiting the disease or condition, i.e., arresting itsdevelopment;

(iii) relieving the disease or condition, i.e., causing regression ofthe disease or condition; or

(iv) relieving the symptoms resulting from the disease or condition,i.e., relieving pain without addressing the underlying disease orcondition. As used herein, the terms “disease” and “condition” may beused interchangeably or may be different in that the particular maladyor condition may not have a known causative agent (so that etiology hasnot yet been worked out) and it is therefore not yet recognized as adisease but only as an undesirable condition or syndrome, wherein a moreor less specific set of symptoms have been identified by clinicians.

The compounds of the invention, or their pharmaceutically acceptablesalts may contain one or more stereocenters and may thus give rise toenantiomers, diastereomers, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present invention is meant to includeall such possible isomers, as well as their racemic and optically pureforms. Optically active (+) and (−), (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, for example, chromatography andfractional crystallization. Conventional techniques for thepreparation/isolation of individual enantiomers include chiral synthesisfrom a suitable optically pure precursor or resolution of the racemate(or the racemate of a salt or derivative) using, for example, chiralhigh pressure liquid chromatography (HPLC). When the compounds describedherein contain olefinic double bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are non-superimposablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds.

Compounds

In an aspect, the invention provides novel lipid compounds which arecapable of combining with other lipid components such as neutral lipids,charged lipids, steroids and/or polymer conjugated-lipids to form lipidnanoparticles with oligonucleotides. Without wishing to be bound bytheory, it is thought that these lipid nanoparticles shieldoligonucleotides from degradation in the serum and provide for effectivedelivery of oligonucleotides to cells in vitro and in vivo.

In one embodiment, the compounds have the following structure (I):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof,wherein:

G¹ is —OH, —NR³R⁴, —(C═O)NR⁵ or —NR³(C═O)R⁵;

G² is —CH₂— or —(C═O)—;

R is, at each occurrence, independently H or OH;

R¹ and R² are each independently branched, saturated or unsaturatedC₁₂-C₃₆ alkyl;

R³ and R⁴ are each independently H or straight or branched, saturated orunsaturated C₁-C₆ alkyl;

R⁵ is straight or branched, saturated or unsaturated C₁-C₆ alkyl; and

n is an integer from 2 to 6.

In some embodiments, R¹ and R² are each independently branched,saturated or unsaturated C₁₂-C₃₀ alkyl, C₁₂-C₂₀ alkyl, or C₁₅-C₂₀ alkyl.In some specific embodiments, R¹ and R² are each saturated. In certainembodiments, at least one of R¹ and R² is unsaturated.

In some of the foregoing embodiments, R¹ and R² have the followingstructure:

In some of the foregoing embodiments, the compound has the followingstructure (IA):

wherein:

R⁶ and R⁷ are, at each occurrence, independently H or straight orbranched, saturated or unsaturated C₁-C₁₄ alkyl;

a and b are each independently an integer ranging from 1 to 15,

provided that R⁶ and a, and R⁷ and b, are each independently selectedsuch that R¹ and R², respectively, are each independently branched,saturated or unsaturated C₁₂-C₃₆ alkyl.

In some of the foregoing embodiments, the compound has the followingstructure (IB):

wherein:

R⁸, R⁹, R¹⁰ and R¹¹ are each independently straight or branched,saturated or unsaturated C₄-C₁₂ alkyl, provided that R⁸ and R⁹, and R¹⁰and R¹¹, are each independently selected such that R¹ and R²,respectively, are each independently branched, saturated or unsaturatedC₁₂-C₃₆ alkyl. In some embodiments of (IB), R⁸, R⁹, R¹⁰ and R¹¹ are eachindependently straight or branched, saturated or unsaturated C₆-C₁₀alkyl. In certain embodiments of (IB), at least one of R⁸, R⁹, R¹⁰ andR¹¹ is unsaturated. In other certain specific embodiments of (IB), eachof R⁸, R⁹, R¹⁰ and R¹¹ is saturated.

In some of the foregoing embodiments, the compound has structure (IA),and in other embodiments, the compound has structure (IB).

In some of the foregoing embodiments, G¹ is —OH, and in some embodimentsG¹ is —NR³R⁴. For example, in some embodiments, G¹ is —NH₂, —NHCH₃ or—N(CH₃)₂. In certain embodiments, G¹ is —(C═O)NR⁵. In certain otherembodiments, G¹ is —NR³(C═O)R⁵. For example, in some embodiments G¹ is—NH(C═O)CH₃ or —NH(C═O)CH₂CH₂CH₃.

In some of the foregoing embodiments, G² is —CH₂—. In some differentembodiments, G² is —(C═O)—.

In some of the foregoing embodiments, n is an integer ranging from 2 to6, for example, in some embodiments n is 2, 3, 4, 5 or 6. In someembodiments, n is 2. In some embodiments, n is 3. In some embodiments, nis 4.

In certain of the foregoing embodiments, at least one of R¹, R², R³, R⁴and R⁵ is unsubstituted. For example, in some embodiments, R¹, R², R³,R⁴ and R⁵ are each unsubstituted. In some embodiments, R³ issubstituted. In other embodiments R⁴ is substituted. In still moreembodiments, R⁵ is substituted. In certain specific embodiments, each ofR³ and R⁴ are substituted. In some embodiments, a substituent on R³, R⁴or R⁵ is hydroxyl. In certain embodiments, R³ and R⁴ are eachsubstituted with hydroxyl.

In some of the foregoing embodiments, at least one R is OH. In otherembodiments, each R is H.

In various different embodiments, the compound has one of the structuresset forth in Table 1 below.

TABLE 1 Representative Compounds No. Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

It is understood that any embodiment of the compounds of structure (I),as set forth above, and any specific substituent and/or variable in thecompound structure (I), as set forth above, may be independentlycombined with other embodiments and/or substituents and/or variables ofcompounds of structure (I) to form embodiments of the inventions notspecifically set forth above. In addition, in the event that a list ofsubstituents and/or variables is listed for any particular R group, Ggroup or variables a, b or n, in a particular embodiment and/or claim,it is understood that each individual substituent and/or variable may bedeleted from the particular embodiment and/or claim and that theremaining list of substituents and/or variables will be considered to bewithin the scope of the invention.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

In some embodiments, lipid nanoparticles comprising a compound ofstructure (I) are provided. The lipid nanoparticles optionally includeexcipients selected from a neutral lipid, a steroid and a polymerconjugated lipid.

In some embodiments, compositions comprising any one or more of thecompounds of structure (I) and a therapeutic agent are provided. Forexample, in some embodiments, the compositions comprise any of thecompounds of structure (I) and a therapeutic agent and one or moreexcipient selected from neutral lipids, steroids and polymer conjugatedlipids. Other pharmaceutically acceptable excipients and/or carriers arealso included in various embodiments of the compositions.

In some embodiments, the neutral lipid is selected from DSPC, DPPC,DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid isDSPC. In various embodiments, the molar ratio of the compound to theneutral lipid ranges from about 2:1 to about 8:1.

In various embodiments, the compositions further comprise a steroid orsteroid analogue. In certain embodiments, the steroid or steroidanalogue is cholesterol. In some of these embodiments, the molar ratioof the compound to cholesterol ranges from about 5:1 to 1:1.

In various embodiments, the polymer conjugated lipid is a pegylatedlipid. For example, some embodiments include a pegylated diacylglycerol(PEG-DAG) such as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), apegylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. Invarious embodiments, the molar ratio of the compound to the pegylatedlipid ranges from about 100:1 to about 20:1.

In some embodiments, the composition comprises a pegylated lipid havingthe following structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

R¹² and R¹³ are each independently a straight or branched, saturated orunsaturated alkyl chain containing from 10 to 30 carbon atoms, whereinthe alkyl chain is optionally interrupted by one or more ester bonds;and

w has a mean value ranging from 30 to 60.

In some embodiments, R¹² and R¹³ are each independently straight,saturated alkyl chains containing from 12 to 16 carbon atoms. In otherembodiments, the average w ranges from about 42 to 55, for example about49.

In some embodiments of the foregoing composition, the therapeutic agentcomprises a nucleic acid. For example, in some embodiments, the nucleicacid is selected from antisense and messenger RNA.

In other different embodiments, the invention is directed to a methodfor administering a therapeutic agent to a patient in need thereof, themethod comprising preparing or providing any of the foregoingcompositions and administering the composition to the patient

For the purposes of administration, embodiments of the compounds of thepresent invention (typically in the form of lipid nanoparticles incombination with a therapeutic agent) may be administered as a rawchemical or may be formulated as pharmaceutical compositions.Pharmaceutical compositions of embodiments of the present inventioncomprise a compound of structure (I) and one or more pharmaceuticallyacceptable carrier, diluent or excipient. In some embodiments, thecompound of structure (I) is present in the composition in an amountwhich is effective to form a lipid nanoparticle and deliver thetherapeutic agent, e.g., for treating a particular disease or conditionof interest. Appropriate concentrations and dosages can be readilydetermined by one skilled in the art.

Administration of the compositions of embodiments of the invention canbe carried out via any of the accepted modes of administration of agentsfor serving similar utilities. The pharmaceutical compositions ofembodiments of the invention may be formulated into preparations insolid, semi-solid, liquid or gaseous forms, such as tablets, capsules,powders, granules, ointments, solutions, suspensions, suppositories,injections, inhalants, gels, microspheres, and aerosols. Typical routesof administering such pharmaceutical compositions include, withoutlimitation, oral, topical, transdermal, inhalation, parenteral,sublingual, buccal, rectal, vaginal, and intranasal. The term parenteralas used herein includes subcutaneous injections, intravenous,intramuscular, intradermal, intrasternal injection or infusiontechniques. Pharmaceutical compositions of embodiments of the inventionare formulated so as to allow the active ingredients contained thereinto be bioavailable upon administration of the composition to a patient.Compositions that will be administered to a subject or patient in someembodiments take the form of one or more dosage units, where forexample, a tablet may be a single dosage unit, and a container of acompound of an embodiments of the invention in aerosol form may hold aplurality of dosage units. Actual methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art; forexample, see Remington: The Science and Practice of Pharmacy, 20thEdition (Philadelphia College of Pharmacy and Science, 2000). In someembodiments, the composition to be administered will, in any event,contain a therapeutically effective amount of a compound of theinvention, or a pharmaceutically acceptable salt thereof, for treatmentof a disease or condition of interest in accordance with the teachingsof this invention.

A pharmaceutical composition of embodiments of the invention may be inthe form of a solid or liquid. In one aspect, the carrier(s) areparticulate, so that the compositions are, for example, in tablet orpowder form. The carrier(s) may be liquid, with the compositions being,for example, an oral syrup, injectable liquid or an aerosol, which isuseful in, for example, inhalatory administration.

When intended for oral administration, the pharmaceutical composition ofcertain embodiments is preferably in either solid or liquid form, wheresemi-solid, semi-liquid, suspension and gel forms are included withinthe forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition of some embodiments may be formulated into a powder,granule, compressed tablet, pill, capsule, chewing gum, wafer or thelike form. Such a solid composition will typically contain one or moreinert diluents or edible carriers. In addition, one or more of thefollowing may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, gum tragacanth or gelatin;excipients such as starch, lactose or dextrins, disintegrating agentssuch as alginic acid, sodium alginate, Primogel, corn starch and thelike; lubricants such as magnesium stearate or Sterotex; glidants suchas colloidal silicon dioxide; sweetening agents such as sucrose orsaccharin; a flavoring agent such as peppermint, methyl salicylate ororange flavoring; and a coloring agent.

When the pharmaceutical composition of some embodiments is in the formof a capsule, for example, a gelatin capsule, it may contain, inaddition to materials of the above type, a liquid carrier such aspolyethylene glycol or oil.

The pharmaceutical composition of some embodiments may be in the form ofa liquid, for example, an elixir, syrup, solution, emulsion orsuspension. The liquid may be for oral administration or for delivery byinjection, as two examples. When intended for oral administration,preferred composition contain, in addition to a compound of structure(I), one or more of a sweetening agent, preservatives, dye/colorant andflavor enhancer. In a composition intended to be administered byinjection, one or more of a surfactant, preservative, wetting agent,dispersing agent, suspending agent, buffer, stabilizer and isotonicagent may be included.

The liquid pharmaceutical compositions of embodiments of the invention,whether they be solutions, suspensions or other like form, may includeone or more of the following adjuvants: sterile diluents such as waterfor injection, saline solution, preferably physiological saline,Ringer's solution, isotonic sodium chloride, fixed oils such assynthetic mono or diglycerides which may serve as the solvent orsuspending medium, polyethylene glycols, glycerin, propylene glycol orother solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose; agents to act ascryoprotectants such as sucrose or trehalose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. Physiological saline is a preferred adjuvant.An injectable pharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of embodiments of the inventionintended for either parenteral or oral administration should contain anamount of a compound of the invention such that a suitable dosage willbe obtained.

The pharmaceutical composition of embodiments of the invention may beintended for topical administration, in which case the carrier maysuitably comprise a solution, emulsion, ointment or gel base. The base,for example, may comprise one or more of the following: petrolatum,lanolin, polyethylene glycols, bee wax, mineral oil, diluents such aswater and alcohol, and emulsifiers and stabilizers. Thickening agentsmay be present in a pharmaceutical composition for topicaladministration. If intended for transdermal administration, thecomposition may include a transdermal patch or iontophoresis device.

The pharmaceutical composition of embodiments of the invention may beintended for rectal administration, in the form, for example, of asuppository, which will melt in the rectum and release the drug. Acomposition for rectal administration may contain an oleaginous base asa suitable nonirritating excipient. Such bases include, withoutlimitation, lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition of embodiments of the invention mayinclude various materials, which modify the physical form of a solid orliquid dosage unit. For example, the composition may include materialsthat form a coating shell around the active ingredients. The materialsthat form the coating shell are typically inert, and may be selectedfrom, for example, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical composition of embodiments of the invention in solidor liquid form may include an agent that binds to the compound of theinvention and thereby assists in the delivery of the compound. Suitableagents that may act in this capacity include a monoclonal or polyclonalantibody, or a protein.

The pharmaceutical composition of embodiments of the invention mayconsist of dosage units that can be administered as an aerosol. The termaerosol is used to denote a variety of systems ranging from those ofcolloidal nature to systems consisting of pressurized packages. Deliverymay be by a liquefied or compressed gas or by a suitable pump systemthat dispenses the active ingredients. Aerosols of compounds ofembodiments of the invention may be delivered in single phase,bi-phasic, or tri-phasic systems in order to deliver the activeingredient(s). Delivery of the aerosol includes the necessary container,activators, valves, subcontainers, and the like, which together may forma kit. One skilled in the art, without undue experimentation, maydetermine preferred aerosols.

The pharmaceutical compositions of embodiments of the invention may beprepared by methodology well known in the pharmaceutical art. Forexample, a pharmaceutical composition intended to be administered byinjection can be prepared by combining the lipid nanoparticles of theinvention with sterile, distilled water or other carrier so as to form asolution. A surfactant may be added to facilitate the formation of ahomogeneous solution or suspension. Surfactants are compounds thatnon-covalently interact with the compound of the invention so as tofacilitate dissolution or homogeneous suspension of the compound in theaqueous delivery system.

The compositions of embodiments of the invention, or theirpharmaceutically acceptable salts, are administered in a therapeuticallyeffective amount, which will vary depending upon a variety of factorsincluding the activity of the specific therapeutic agent employed; themetabolic stability and length of action of the therapeutic agent; theage, body weight, general health, sex, and diet of the patient; the modeand time of administration; the rate of excretion; the drug combination;the severity of the particular disorder or condition; and the subjectundergoing therapy.

Compositions of embodiments of the invention may also be administeredsimultaneously with, prior to, or after administration of one or moreother therapeutic agents. Such combination therapy includesadministration of a single pharmaceutical dosage formulation of acomposition of embodiments of the invention and one or more additionalactive agents, as well as administration of the composition ofembodiments of the invention and each active agent in its own separatepharmaceutical dosage formulation. For example, a composition ofembodiments of the invention and the other active agent can beadministered to the patient together in a single oral dosage compositionsuch as a tablet or capsule, or each agent administered in separate oraldosage formulations. Where separate dosage formulations are used, thecompounds of embodiments of the invention and one or more additionalactive agents can be administered at essentially the same time, i.e.,concurrently, or at separately staggered times, i.e., sequentially;combination therapy is understood to include all these regimens.

Preparation methods for the above compounds and compositions aredescribed herein below and/or known in the art.

It will be appreciated by those skilled in the art that in the processdescribed herein the functional groups of intermediate compounds mayneed to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999),3^(rd) Ed., Wiley. As one of skill in the art would appreciate, theprotecting group may also be a polymer resin such as a Wang resin, Rinkresin or 2-chlorotrityl-chloride resin.

It will also be appreciated by those skilled in the art, although suchprotected derivatives of compounds of this invention may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of theinvention which are pharmacologically active. Such derivatives maytherefore be described as “prodrugs”. All prodrugs of compounds of thisinvention are included within the scope of the invention.

Furthermore, compounds of embodiments of the invention which exist infree base or acid form can be converted to their pharmaceuticallyacceptable salts by treatment with the appropriate inorganic or organicbase or acid by methods known to one skilled in the art. Salts ofcompounds of embodiments of the invention can be converted to their freebase or acid form by standard techniques.

The following General Reaction Scheme 1 illustrates exemplary methods tomake compounds of this invention, i.e., compounds of structure (I):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein R¹, R², G¹, G², and n are as defined herein. It is understoodthat one skilled in the art may be able to make these compounds bysimilar methods or by combining other methods known to one skilled inthe art. It is also understood that one skilled in the art would be ableto make, in a similar manner as described below, other compounds ofstructure (I) not specifically illustrated below by using theappropriate starting components and modifying the parameters of thesynthesis as needed. In general, starting components may be obtainedfrom sources such as Sigma Aldrich, Lancaster Synthesis, Inc.,Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. orsynthesized according to sources known to those skilled in the art (see,for example, Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th edition (Wiley, December 2000)) or prepared as describedin this invention.

General Reaction Scheme I provides an exemplary method for preparationof compounds of structure (I). G¹ and n in General reaction Scheme 1 areas defined herein, and R^(1′) refers to a one-carbon shorter homologueof R¹. Compounds of structure A-1 are purchased or prepared according tomethods known in the art. Reaction of A-1 under appropriate oxidationconditions (e.g., TEMPO) yields aldehyde A-2, which can then undergo areductive amination with A-3 using an appropriate reagent (e.g., sodiumtriacetoxyborohydride) to yield a compound of structure (I).

It should be noted that various alternative strategies for preparationof compounds of structure (I) are available to those of ordinary skillin the art. For example, other compounds of structure (I) wherein can beprepared according to analogous methods using the appropriate startingmaterial. Further, General Reaction Scheme 1 depicts preparation of acompound of structure (I), wherein R¹ and R² are the same; however, thisis not a required aspect of the invention and modifications to the abovereaction scheme are possible to yield compounds wherein R¹ and R² aredifferent. The use of protecting groups as needed and other modificationto the above General Reaction Scheme will be readily apparent to one ofordinary skill in the art.

The following examples are provided for purpose of illustration and notlimitation.

Example 1 Luciferase mRNA In Vivo Evaluation Using the LipidNanoparticle Compositions

Lipid nanoparticles are prepared and tested according to the generalprocedures described in PCT Pub. Nos. WO 2015/199952 and WO 2017/004143,the full disclosures of which are incorporated herein by reference.Briefly, cationic lipid, DSPC, cholesterol and PEG-lipid are solubilizedin ethanol at a molar ratio of about 50:10:38.5:1.5 or about47.5:10:40.8:1.7. Lipid nanoparticles (LNP) are prepared at a totallipid to mRNA weight ratio of approximately 10:1 to 30:1. The mRNA isdiluted to 0.2 mg/mL in 10 to 50 mM citrate or acetate buffer, pH 4.Syringe pumps are used to mix the ethanolic lipid solution with the mRNAaqueous solution at a ratio of about 1:5 to 1:3 (vol/vol) with totalflow rates above 15 mL/min. The ethanol is then removed and the externalbuffer replaced with PBS by dialysis. Finally, the lipid nanoparticlesare filtered through a 0.2 μm pore sterile filter. Lipid nanoparticleparticle size is approximately 55-95 nm diameter, and in some instancesapproximately 70-90 nm diameter as determined by quasi-elastic lightscattering using a Malvern Zetasizer Nano ZS (Malvern, UK).

Studies are performed in 6-8 week old female C57BL/6 mice (CharlesRiver) or 8-10 week old CD-1 (Harlan) mice (Charles River) according toguidelines established by an institutional animal care committee (ACC)and the Canadian Council on Animal Care (CCAC). Varying doses ofmRNA-lipid nanoparticle are systemically administered by tail veininjection and animals euthanized at a specific time point (e.g., 4hours) post-administration. Liver and spleen are collected inpre-weighed tubes, weights determined, immediately snap frozen in liquidnitrogen and stored at −80° C. until processing for analysis.

For liver, approximately 50 mg is dissected for analyses in a 2 mLFastPrep tubes (MP Biomedicals, Solon OH). ¼″ ceramic sphere (MPBiomedicals) is added to each tube and 500 μL of Glo Lysis Buffer—GLB(Promega, Madison Wis.) equilibrated to room temperature is added toliver tissue. Liver tissues are homogenized with the FastPrep24instrument (MP Biomedicals) at 2×6.0 m/s for 15 seconds. Homogenate isincubated at room temperature for 5 minutes prior to a 1:4 dilution inGLB and assessed using SteadyGlo Luciferase assay system (Promega).Specifically, 50 μL of diluted tissue homogenate is reacted with 50 μLof SteadyGlo substrate, shaken for 10 seconds followed by 5 minuteincubation and then quantitated using a CentroXS³ LB 960 luminometer(Berthold Technologies, Germany). The amount of protein assayed isdetermined by using the BCA protein assay kit (Pierce, Rockford Ill.).Relative luminescence units (RLU) are then normalized to total ugprotein assayed. To convert RLU to ng luciferase a standard curve isgenerated with QuantiLum Recombinant Luciferase (Promega).

The FLuc mRNA (L-6107 or L-7202) from Trilink Biotechnologies willexpress a luciferase protein, originally isolated from the firefly,photinus pyralis. FLuc is commonly used in mammalian cell culture tomeasure both gene expression and cell viability. It emitsbioluminescence in the presence of the substrate, luciferin. This cappedand polyadenylated mRNA is fully substituted with respect to uridineand/or cytidine nucleosides.

Example 2 Determination of pK_(A) of Formulated Lipids

As described elsewhere, the pKa of formulated cationic lipids iscorrelated with the effectiveness of LNPs for delivery of nucleic acids(see Jayaraman et al, Angewandte Chemie, International Edition (2012),51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176(2010)). The preferred range of pKa is ˜5 to ˜7. The pK_(a) of eachcationic lipid is determined in lipid nanoparticles using an assay basedon fluorescence of 2-(p-toluidino)-6-napthalene sulfonic acid (TNS).Lipid nanoparticles comprising cationic lipid/DSPC/cholesterol/PEG-lipid(50/10/38.5/1.5 mol %) in PBS at a concentration of 0.4 mM total lipidare prepared using the in-line process as described in Example 1. TNS isprepared as a 100 μM stock solution in distilled water. Vesicles arediluted to 24 μM lipid in 2 mL of buffered solutions containing, 10 mMHEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where the pHranged from 2.5 to 11. An aliquot of the TNS solution is added to give afinal concentration of 1 μM and following vortex mixing fluorescenceintensity is measured at room temperature in a SLM Aminco Series 2Luminescence Spectrophotometer using excitation and emission wavelengthsof 321 nm and 445 nm. A sigmoidal best fit analysis is applied to thefluorescence data and the pK_(a) was measured as the pH giving rise tohalf-maximal fluorescence intensity.

Example 3 Determination of Efficacy of Lipid Nanoparticle FormulationsContaining Various Cationic Lipids Using an In Vivo Luciferase mRNAExpression Rodent Model

The cationic lipids shown in Table 2 have previously been tested withnucleic acids. For comparative purposes, these lipids were also used toformulate lipid nanoparticles containing the FLuc mRNA (L-6107) using anin line mixing method, as described in Example 1 and in PCT/US10/22614,which is hereby incorporated by reference in its entirety. Lipidnanoparticles were formulated using the following molar ratio: 50%Cationic lipid/10% distearoylphosphatidylcholine (DSPC)/38.5%Cholesterol/1.5% PEG lipid (“PEG-DMG”, i.e.,1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol, with anaverage PEG molecular weight of 2000). In alternate embodiments,cationic lipid, DSPC, cholesterol and PEG-lipid are formulated at amolar ratio of approximately 47.5:10:40.8:1.7. Relative activity wasdetermined by measuring luciferase expression in the liver 4 hoursfollowing administration via tail vein injection as described inExample 1. The activity was compared at a dose of 0.3 and 1.0 mg mRNA/kgand expressed as ng luciferase/g liver measured 4 hours afteradministration, as described in Example 1.

TABLE 2 Comparator Lipids showing activity with mRNA Liver Luc Liver Luc@ 0.3 mg/ @ 1.0 mg/ Compound kg dose kg dose Structure MC2  4 ± 1  N/D

DLinDMA  13 ± 3   67 ± 20 

MC4  41 ± 10  N/D

XTC2  80 ± 28  237 ± 99 

MC3 198 ± 126 757 ± 528

319 (2% PEG) 258 ± 67  681 ± 203

137 281 ± 203 588 ± 303

A  77 ± 40  203 ± 122

Representative compounds of the invention shown in Table 3 wereformulated using the following molar ratio: 50% cationic lipid/10%distearoylphosphatidylcholine (DSPC)/38.5% Cholesterol/1.5% PEG lipid(“PEG-DMA”2-[2-(ω-methoxy(polyethyleneglycol₂₀₀₀)ethoxy]-N,N-ditetradecylacetamide)or 47.5% cationic lipid/10% DSPC/40.8% Cholesterol/1.7% PEG lipid.Relative activity was determined by measuring luciferase expression inthe liver 4 hours following administration via tail vein injection asdescribed in Example 1. The activity was compared at a dose of 0.3, 0.5and/or 1.0 mg mRNA/kg and expressed as ng luciferase/g liver measured 4hours after administration, as described in Example 1.

Compound numbers in Table 3 refer to the compound numbers of Table 1.

TABLE 3 Novel Cationic Lipids and Associated Activity Liver Luc LiverLuc Liver Luc Cmp. @ 0.3 mg/kg (ng @ 0.5 mg/kg @ 1.0 mg/kg No. pK_(a)luc/g liver) (ng luc/g liver) (ng luc/g liver) 4 5.55 87 ± 11 n/a  233 ±121 5 5.77 2840 ± 757  n/a 13739 ± 7631 15 5.91 n/a 2417 ± 833  n/a 165.87 n/a 52 ± 26 n/a 17 6.49 n/a 12 ± 5  n/a

Example 4 Synthesis of 2-Hexyldecanal

A solution of 2-hexyldecanol (12.8 g) in dichloromethane (30 mL) wastreated with potassium bromide solution (0.96 g in 3.8 mL water) and(2,2,6,6-tetramethylpiperidin-1-yl)oyl oroxidanyl (TEMPO, 80 mg). Thesolution was cooled in an ice/salt mixture for 15 minutes. A solution ofconcentrated sodium hypochlorite (40 mL of 11-15% sodium hypochloritewith 15 mL of saturated aqueous sodium bicarbonate) was slowly addeddropwise to the reaction mixture. The reaction was stirred for 15minutes and then extracted between hexane and water. The crude productwas passed down a silica gel column using hexane as the eluent.

Example 5 Synthesis of Compound 1

A solution of 2-hexyldecanal (4.3 g) in dichloromethane (20 mL) wastreated with 4-hydroxybutylamine (0.41 g), acetic acid (0.58 g) andsodium triacetoxyborohydride (2.10 g). The reaction was stirredovernight and then washed with aqueous sodium bicarbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed in vacuo. The residue was passed down a silica gelcolumn using initially 2% acetic acid/dichloromethane, followed by a2-8% methanol/dichloromethane gradient. The purified product wasdissolved in hexane and washed with sodium hydrogen carbonate solution.The solvent was removed and the residue dissolved in ˜5 mL hexane. Thesolution was passed through a silica gel plug, and dried under anitrogen stream, yielding 0.74 g of the desired product.

Example 6 Synthesis of Compound 2

A solution of 2-hexyldecanal (4.3 g) in dichloromethane (20 mL) wastreated with 2-N,N-dimethylaminoethylamine (0.40 g), acetic acid (0.59g) and sodium triacetoxyborohydride (2.10 g). The reaction was stirredfor two hours and then washed with aqueous sodium bicarbonate solution.The organic phase was dried over anhydrous magnesium sulfate, filteredand dried in vacuo. The residue was passed down a silica gel columnusing initially 2% acetic acid/dichloromethane, followed by a 2-8%methanol/dichloromethane gradient. The purified product was dissolved inhexane and washed with sodium hydrogen carbonate solution. The solventwas removed from the organic fraction and the residue dissolved in ˜5 mLhexane. The solution was passed through a silica gel plug, and driedunder a nitrogen stream, yielding 1.20 g of the desired product.

Example 7 Synthesis of Compound 3

A solution of 2-hexyldecanal (4.0 g) in dichloromethane (20 mL) wastreated with 3-N,N-dimethylaminopropylamine (0.43 g), acetic acid (0.58g) and sodium triacetoxyborohydride (2.05 g). The reaction was stirredovernight and then washed with aqueous sodium bicarbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered anddried in vacuo. The residue was passed down a silica gel column usinginitially 2% acetic acid/dichloromethane, followed by a 2-8%methanol/dichloromethane gradient. The purified product was dissolved inhexane and washed with sodium hydrogen carbonate solution. The solventwas removed from the organic fraction and the residue dissolved in ˜5 mLhexane. The solution was passed through a silica gel plug, and driedunder a nitrogen stream, yielding 0.74 g of the desired product.

Example 8 Synthesis of Compound 4

A solution of 2-hexyldecanal (3.1 g) in dichloromethane (20 mL) wastreated with 4-N,N-dimethylaminobutylamine (0.50 g), acetic acid (0.57g) and sodium triacetoxyborohydride (2.7 g). The reaction was stirredovernight and then washed with aqueous sodium bicarbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered anddried in vacuo. The residue was passed down a silica gel column usinginitially 2% acetic acid/dichloromethane, followed by a 1-2%methanol/dichloromethane gradient. The purified product was dissolved inhexane and washed with sodium hydrogen carbonate solution. The solventwas removed from the organic fraction and the residue dissolved in ˜2 mLhexane. The solution was passed through a silica gel plug, and driedunder a nitrogen stream, yielding 1.75 g of compound 4.

Example 9 Synthesis of 2-hexyldecanoylamide

A solution of 2-hexyldecanoic acid (26 g) in benzene (30 mL) was treatedwith oxalyl chloride (15 mL). The reaction was stirred until gasevolution ceased, after which the solvent was removed on a rotovap andthe residue dried under vacuum for 4 hours. The crude 2-hexyldecanoylchloride was dissolved in dichloromethane (100 mL) and added slowly to astirred solution of concentrated ammonium hydroxide (150 mL). Thereaction mixture was allowed to stand for two hours and the aqueoussupernatant decanted off. The organic phase was washed twice with waterin the same manner, then filtered. The collected precipitate was dried,yielding crude 2-hexyldecanoylamide (22 g) as a white powder.

Example 10 Synthesis of 2-hexyldecanylamine

A suspension of 2-hexyldecanoylamide (8.2 g) in dry tetrahydrofuran (40mL) was treated with lithium aluminium hydride (1.1 g, added slowly).The reaction was stirred for two hours and then excess methanol wasslowly added. Dichloromethane (150 mL) was added, followed by water (2mL). The reaction mixture was filtered and the solvent removed from thefiltrate, yielding crude 2-hexyldecanylamine (4.7 g).

Example 11 Synthesis of N-(2′-hexyldecanyl)-2-hexyldecanoylamide

A solution of 2-hexyldecanylamine (4.7 g) in dichloromethane (100 mL)was treated with 2-hexyldecanoyl chloride (5.5 g, dissolved in 50 mLdichloromethane), followed by triethylamine (4 mL). The reaction wasstirred for an hour and then washed with dilute hydrochloric acid. Theorganic phase was dried over anhydrous magnesium sulfate, filtered, andthe solvent removed. The residue (˜10 g) was combined with the crudeproducts from a second reaction (˜7 g) and passed down a silica gel (100g) using a 0-10% methanol/dichloromethane gradient, yieldingN-(2′-hexyldecanyl)-2-hexyldecanoylamide (14.4 g).

Example 12 Synthesis of Di-(2-Hexyldecanyl)amine

A solution of N-(2′-hexyldecanyl)-2-hexyldecanoylamide (14.4 g) in drytetrahydrofuran (100 mL) was treated with lithium aluminum hydride (2 g)and refluxed overnight. Excess methanol was slowly added to destroyexcess reducing agent. Dichloromethane (200 mL) was added, followed bywater (2 mL). The mixture was then filtered and the solvent removed. Theresidue was suspended in hexane, filtered, and the solvent removed. Theresidue was passed down a silica gel (100 g) column using 2% aceticacid/dichloromethane, followed by a 2-16% methanol/dichloromethanegradient. Purified fractions were washed between hexane and aqueoussodium bicarbonate solution. Removal of the solvent yieldeddi-(2-hexyldecanyl)amine as a colorless oil (9.5 g).

Example 13 Synthesis of

A solution of di-(2-hexyldecanyl)amine (1.4 g) in dichloromethane (20mL) was treated with triethylamine (1 mL) and a solution of5-bromopentanoyl chloride (2 g) in dichloromethane (20 mL). The reactionwas stirred for an hour and then washed with dilute aqueous hydrochloricacid. The organic phase was dried over anhydrous magnesium sulfate,filtered and the solvent removed. The residue was dissolved in a 2Msolution of dimethylamine in tetrahydrofuran (30 mL) and stirredovernight. Most of the solvent was removed and the residue partitionedbetween hexane and dilute aqueous hydrochloric acid. The organic phasewas washed with water, dried over anhydrous magnesium sulfate, filteredand the solvent removed. The residue was passed down a silica gel columnusing 2% acetic acid/dichloromethane, followed by a 2-12%methanol/dichloromethane gradient, yielding the target compound (0.95 g)as a colorless oil.

Example 14 Synthesis of Compound 5

A solution of

(0.3 g) in tetrahydrofuran (20 mL) was treated with lithium aluminumhydride (0.2 g, added slowly). The reaction was stirred overnight.Excess methanol was then slowly added, followed by dichloromethane (100mL). The suspension was filtered and the solvent removed. The residuewas suspended in dichloromethane, filtered again, and the solventremoved. The crude product was passed down a silica gel column using 2%acetic acid/dichloromethane followed by a 2-16% methanol/dichloromethanegradient. The purified fractions were partitioned between hexane andaqueous sodium bicarbonate solution. The solvent was removed, yieldingcompound 5 (0.11 g) as a colorless oil.

Example 15 Synthesis of Compound 13

A solution of 2-hexyldecanal (3.0 g) in dichloromethane (30 mL) wastreated with 3-aminopropan-1,2-diol (0.36 g) and sodiumtriacetoxyborohydride (2.6 g). The reaction was stirred for three daysand then washed with aqueous sodium bicarbonate solution. The organicphase was dried over anhydrous magnesium sulfate, filtered and dried invacuo. The residue was passed down a silica gel column using initially2% acetic acid/dichloromethane, followed by a 2-6%methanol/dichloromethane gradient. The purified product was dissolved inhexane and washed with sodium hydrogen carbonate solution. The solventwas removed from the organic fraction and the residue dissolved in ˜5 mLhexane. The solution was passed through a silica gel plug, and driedunder a nitrogen stream, yielding compound 13 (1.75 g) as a colorlessoil.

Example 16 Synthesis of

A solution of di-(2-hexyldecanyl)amine (1.5 g) in dichloromethane (10mL) was treated with triethylamine (20 drops) and a solution of6-bromohexanoyl chloride (1 g) in dichloromethane (10 mL). The reactionwas stirred for thirty minutes and then the solvent was removed on arotovap. The residue was dissolved in dichloromethane, filtered througha silica gel bed and the solvent removed. The residue was dissolved in a2M solution of dimethylamine in tetrahydrofuran (30 mL) and stirredovernight. Most of the solvent was removed and the residue partitionedbetween dichloromethane and aqueous sodium bicarbonate solution. Theorganic phase was washed with water, dried over anhydrous magnesiumsulfate, filtered and the solvent removed. The residue was passed down asilica gel column using a 0-8% methanol/dichloromethane gradient,yielding the target compound (0.26 g) as a colorless oil.

Example 17 Synthesis of

A solution of di-(2-hexyldecanyl)amine (1.25 g) in dichloromethane (20mL) was treated with triethylamine (5 mL) and 5-bromopentanoyl chloride(1 g). The reaction was stirred for 10 minutes, filtered and the solventremoved. The residue was dissolved in dichloromethane and washed withdilute aqueous hydrochloric acid. The organic phase was dried overanhydrous magnesium sulfate, filtered and the solvent removed. Theresidue was treated with a solution of diethanolamine (4.9 g) intetrahydrofuran (20 mL) and heated to 45 C overnight. The reactionmixture was washed twice with water, and the solvent removed. Theresidue was passed down a silica gel column using a 0-12%methanol/dichloromethane gradient, yielding the target compound (0.32 g)as a colorless oil.

Example 18 Synthesis of Compound 14

A solution of

(0.52 g) in tetrahydrofuran (10 mL) was treated with lithium aluminumhydride (0.50 g, added slowly). The reaction was stirred overnight.Excess methanol was then slowly added, followed by dichloromethane (100mL) and water (1 mL). The suspension was filtered and the solventremoved. The residue was suspended in dichloromethane, filtered again,and the solvent removed. The crude product was passed down a silica gelcolumn using a 1-12% methanol/dichloromethane gradient yielding compound14 (0.12 g) as a colorless oil.

Example 19 Synthesis of Compound 15

A solution of

(0.67 g) in tetrahydrofuran (20 mL) was treated with lithium aluminumhydride (0.35 g, added slowly). The reaction was stirred for two hours.Excess methanol was then slowly added, followed by dichloromethane (100mL) and water (2 mL). The suspension was filtered and the solventremoved. The crude product was passed down a silica gel column using a0-12% methanol/dichloromethane gradient yielding compound 15 (0.30 g) asa colorless oil.

Example 20 Synthesis of

A solution of di-(2-hexyldecanyl)amine (1.25 g) in dichloromethane (20mL) was treated with triethylamine (5 mL) and 5-bromopentanoyl chloride(1 g). The reaction was stirred for 10 minutes, filtered and the solventremoved. The residue was dissolved in dichloromethane and washed withdilute aqueous hydrochloric acid. The organic phase was dried overanhydrous magnesium sulfate, filtered and the solvent removed. Theresidue was treated with isopropylamine (20 mL) and stirred overnight.The solvent was removed and the residue washed between hexane andaqueous sodium bicarbonate solution. The solvent was removed and residuepassed down a silica gel column using a 0-8% methanol/dichloromethanegradient, yielding the target compound (0.67 g) as a colorless oil.

Example 21 Synthesis of Compound 16

A solution of

(0.22 g) in tetrahydrofuran (10 mL) was treated with lithium aluminumhydride (0.25 g, added slowly). The reaction was stirred overnight.Excess methanol was then slowly added, followed by dichloromethane (50mL) and water (1 mL). The suspension was filtered and the solventremoved. The residue was suspended in dichloromethane, filtered again,and the solvent removed, yielding 0.24 g of crude product. This wascombined with the products from a second reaction for a total of 0.42 g.The crude product was passed down a silica gel column using a 0-12%methanol/dichloromethane gradient. The purified fractions werepartitioned between hexane and aqueous sodium bicarbonate solution. Thesolvent was removed, yielding compound 16 (0.24 g) as a colorless oil.

Example 22 Synthesis of Compound 17

A solution of di-(2-hexyldecanyl)amine (1.1 g) in hexane (20 mL) wastreated with 3-bromopropionoyl chloride (0.8 g) and triethylamine (2mL), and stirred for two hours. The solution was washed with water andthe solvent removed. The residue was dissolved in a 2M solution ofdimethylamine in tetrahydrofuran (15 ml) and stirred overnight. Thesolvent was removed and the residue passed down a silica gel columnusing an acetic acid/methanol/dichloromethane (2-0%; 0-16%; 98-84%)gradient. The purified product was dissolved in tetrahydrofuran (20 mL)and treated with lithium aluminum hydride (0.6 g) overnight. Excessmethanol was slowly added, followed by dichloromethane (50 mL) and water(1 mL). The suspension was filtered and the solvent removed. The residuewas passed down a silica gel column using an aceticacid/methanol/dichloromethane (2-0%; 0-16%; 98-84%) gradient, yieldingcompound 17 (0.27 g) as a colorless oil.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, if any,including U.S. Provisional Patent Application No. 62/485,278, filed Apr.13, 2017, are incorporated herein by reference, in their entirety.Aspects of the embodiments can be modified, if necessary to employconcepts of the various patents, applications and publications toprovide yet further embodiments. These and other changes can be made tothe embodiments in light of the above-detailed description. In general,in the following claims, the terms used should not be construed to limitthe claims to the specific embodiments disclosed in the specificationand the claims, but should be construed to include all possibleembodiments along with the full scope of equivalents to which suchclaims are entitled. Accordingly, the claims are not limited by thedisclosure.

1. A compound having the following structure (I):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein: G¹ is —OH, —NR³R⁴, —(C═O)NR⁵ or —NR³(C═O)R⁵; G² is —CH₂— or—(C═O)—; R is, at each occurrence, independently H or OH; R¹ and R² areeach independently optionally substituted branched, saturated orunsaturated C₁₂-C₃₆ alkyl; R³ and R⁴ are each independently H oroptionally substituted straight or branched, saturated or unsaturatedC₁-C₆ alkyl; R⁵ is optionally substituted straight or branched,saturated or unsaturated C₁-C₆ alkyl; and n is an integer from 2 to 6.2. The compound of claim 1, having the following structure (IA):

wherein: R⁶ and R⁷ are, at each occurrence, independently H or straightor branched, saturated or unsaturated C₁-C₁₄ alkyl; a and b are eachindependently an integer ranging from 1 to 15, provided that R⁶ and a,and R⁷ and b, are each independently selected such that R¹ and R²,respectively, are each independently branched, saturated or unsaturatedC₁₂-C₃₆ alkyl.
 3. The compound of any one of claims 1 or 2, having thefollowing structure (IB):

wherein R⁸, R⁹, R¹⁰ and R¹¹ are each independently straight or branched,saturated or unsaturated C₄-C₁₂ alkyl, provided that R⁸ and R⁹, and R¹⁰and R¹¹, are each independently selected such that R¹ and R²,respectively, are each independently branched, saturated or unsaturatedC₁₂-C₃₆ alkyl.
 4. The compound of claim 3, wherein R⁸, R⁹, R¹⁰ and R¹¹are each independently straight or branched, saturated or unsaturatedC₆-C₁₀ alkyl.
 5. The compound of any one of claims 3 or 4, wherein atleast one of R⁸, R⁹, R¹⁰ and R¹¹ is unsaturated.
 6. The compound of anyone of claims 3 or 4, wherein each of R⁸, R⁹, R¹⁰ and R¹¹ is saturated.7. The compound of claim 1, wherein R¹ and R² are each independentlybranched, saturated or unsaturated C₁₂-C₃₀ alkyl.
 8. The compound ofclaim 1, wherein R¹ and R² are each independently branched, saturated orunsaturated C₁₂-C₂₀ alkyl.
 9. The compound of claim 1, wherein R¹ and R²are each independently branched, saturated or unsaturated C₁₅-C₂₀ alkyl.10. The compound of any one of claims 1 or 7-9, wherein R¹ and R² areeach saturated.
 11. The compound of any one of claims 1 or 7-9, whereinat least one of R¹ and R² is unsaturated.
 12. The compound of claim 1,wherein R¹ and R² have the following structure:


13. The compound of any one of claims 1-12, wherein G¹ is —OH.
 14. Thecompound of any one of claims 1-12, wherein G¹ is —NR³R⁴.
 15. Thecompound of claim 14, wherein G¹ is —NH₂, —NHCH₃ or —N(CH₃)₂.
 16. Thecompound of any one of claims 1-12, wherein G¹ is —(C═O)NR⁵.
 17. Thecompound of any one of claims 1-12, wherein G¹ is —NR³(C═O)R⁵.
 18. Thecompound of claim 17, wherein G¹ is —NH(C═O)CH₃ or —NH(C═O)CH₂CH₂CH₃.19. The compound of any one of claims 1-18, wherein G² is —CH₂—.
 20. Thecompound of any one of claims 1-18, wherein G² is —(C═O)—.
 21. Thecompound of any one of claims 1-20, wherein n is
 2. 22. The compound ofany one of claims 1-21, wherein n is
 3. 23. The compound of any one ofclaims 1-21, wherein n is
 4. 24. The compound of any one of claims 1-23,wherein R¹, R², R³, R⁴ and R⁵ are each unsubstituted.
 25. The compoundof any one of claims 1-23, wherein R³ and R⁴ are each independentlysubstituted.
 26. The compound of claim 25, wherein R³ and R⁴ are eachsubstituted with hydroxyl.
 27. The compound of any one of claims 1-26,wherein at least one R is OH.
 28. The compound of any one of claims1-26, wherein each R is H.
 29. The compound of claim 1, having one ofthe following structures:


30. A composition comprising the compound of any one of claims 1-29 anda therapeutic agent.
 31. The composition of claim 30, further comprisingone or more excipient selected from neutral lipids, steroids and polymerconjugated lipids.
 32. The composition of claim 31, wherein thecomposition comprises one or more neutral lipids selected from DSPC,DPPC, DMPC, DOPC, POPC, DOPE and SM.
 33. The composition of claim 32,wherein the neutral lipid is DSPC.
 34. The composition of any one ofclaims 30-33, wherein the molar ratio of the compound to the neutrallipid ranges from about 2:1 to about 8:1.
 35. The composition of any oneof claims 31-34, wherein the steroid is cholesterol.
 36. The compositionof claim 35, wherein the molar ratio of the compound to cholesterolranges from 5:1 to 1:1.
 37. The composition of any one of claims 31-36,wherein the polymer conjugated lipid is pegylated lipid.
 38. Thecomposition of claim 37, wherein the molar ratio of the compound topegylated lipid ranges from about 100:1 to about 20:1.
 39. Thecomposition of anyone of claims 37 or 38, wherein the pegylated lipid isPEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or a PEG dialkyoxypropylcarbamate.40. The composition of any one of claims 37 or 38, wherein the pegylatedlipid has the following structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein: R¹² and R¹³ are each independently a straight or branched,saturated or unsaturated alkyl chain containing from 10 to 30 carbonatoms, wherein the alkyl chain is optionally interrupted by one or moreester bonds; and w has a mean value ranging from 30 to
 60. 41. Thecomposition of claim 40, wherein R¹² and R¹³ are each independentlystraight, saturated alkyl chains containing from 12 to 16 carbon atoms.42. The composition of any one of claims 40 or 41, wherein the average wis about
 49. 43. The composition of any one of claims 30-42, wherein thetherapeutic agent comprises a nucleic acid.
 44. The composition of claim43, wherein the nucleic acid is selected from antisense and messengerRNA.
 45. A method for administering a therapeutic agent to a patient inneed thereof, the method comprising preparing or providing thecomposition of any one of claims 30-44, and administering thecomposition to the patient.